Plants which synthesize a modified starch, process for the production thereof and modified starch

ABSTRACT

Nucleic acid molecules are described encoding a starch granule-bound protein as well as methods and recombinant DNA molecules for the production of transgenic plant cells and plants synthesizing a modified starch with modified viscosity properties and a modified phosphate content. Moreover, the plant cells and plants resulting from those methods as well as the starch obtainable therefrom are described.

The present invention relates to nucleic acid molecules encoding astarch granule-bound protein as well as to methods and recombinant DNAmolecules for the production of transgenic plant cells and plantssynthesizing a modified starch with modified properties of viscosity anda modified phosphate content. The invention also relates to thetransgenic plant cells and plants resulting from these methods and tothe starch obtainable from the transgenic plant cells and plants.

The polysaccharide starch, which constitutes one of the most importantstorage substances in plants, is not only used in the area of foodstuffsbut also plays a significant role as a regenerative material in themanufacturing of industrial products. In order to enable the use of thisraw material in as many areas as possible, it is necessary to obtain alarge variety of substances as well as to adapt these substances to thevarying demands of the processing industry.

Although starch consists of a chemically homogeneous basic component,namely glucose, it does not constitute a homogeneous raw material. It israther a complex mixture of various types of molecules which differ fromeach other in their degree of polymerization and in the degree ofbranching of the glucose chains. One differentiates particularly betweenamylose-starch, a basically non-branched polymer made up ofα-1,4-glycosidically branched glucose molecules, and amylopectin-starchwhich in turn is a mixture of more or less heavily branched glucosechains. The branching results from the occurrence of α-1,6-glycosidicinterlinkings.

The molecular structure of starch which is mainly determined by itsdegree of branching, the amylose/amylopectin ratio, the averagechain-length and the occurrence of phosphate groups is significant forimportant functional properties of starch or, respectively, its aqueoussolutions. Important functional properties are for example solubility ofthe starch, tendency to retrogradation, capability of film formation,viscosity, colour stability, pastification properties, i.e. binding andgluing properties, as well as cold resistance. The starch granule sizemay also be significant for the various uses. The production of starchwith a high amylose content is particularly significant. Furthermore,modified starch contained in plant cells may, finder certain conditions,favorably alter the behavior of the plant cell. For example, it would bepossible to decrease the starch degradation during the storage of thestarch-containing organs such as seeds and tubers prior to their furtherprocessing by, for example, starch extraction. Moreover, there is someinterest in producing modified starches which would render plant cellsand plant organs containing this starch more suitable for furtherprocessing, such as for the production of popcorn or corn flakes frompotato or of French fries, crisps or potato powder from potatoes. Thereis a particular interest in improving the starches in such a way, thatthey show a reduced “cold sweetening”, i.e. a decreased release ofreduced sugars (especially glucose) during long-term storage at lowtemperatures. Specifically potatoes are often stored at temperatures of4-8° C. in order to minimize the degradation of starch during storage.The reducing sugars released thereby, in particular glucose, lead toundesired browning reactions (so-called Maillard reactions) in theproduction of French fries and crisps.

Starch which can be isolated from plants is often adapted to certainindustrial purposes by means of chemical modifications which are usuallytime-consuming and expensive. Therefore it is desirable to findpossibilities to produce plants synthesizing a starch the properties ofwhich already meet the demands of the processing industry.

Conventional methods for producing such plants are classical breedingmethods and the production of mutants. Thus, for example, a mutant wasproduced from maize synthesizing starch with an altered viscosity (U.S.Pat. No. 5,331,108) and a maize variety (waxy maize) was established bymeans of breeding the starch of which consists of almost 100%amylopectin (Akasuka and Nelson, J. Biol. Chem. 241 (1966), 2280-2285).Furthermore, mutants of potato and pea have been described whichsynthesize starches with a high amylose content (70% in maize or up to50% in pea). These mutants have so far not been characterized on themolecular level and therefore do not allow for the production ofcorresponding mutants in other starch-storing plants.

Alternatively, plants synthesizing starch with altered properties may beproduced by means of recombinant DNA techniques. In various cases, forexample, the recombinant modification of potato plants aiming ataltering the starch synthesized in these plants has been described (e.g.WO 92/11376; WO 92/14827). However, in order to make use of recombinantDNA techniques, DNA sequences are required the gene products of whichinfluence starch synthesis, starch modification or starch degradation.

Therefore, the problem underlying the present invention is to providenucleic acid molecules and methods which allow for the alteration ofplants in such a way, that they synthesize a starch which differs fromstarch naturally synthesized in plants with respect to its physicaland/or chemical properties, in particular a highly amylose-containingstarch, and is therefore more suitable for general and/or particularuses.

This problem is solved by the provision of the embodiments described inthe claims.

Therefore, the present invention relates to nucleic acid moleculesencoding a protein with the amino acid sequence indicated in Seq ID No.2. Such proteins are present in the plastids of plant cells, bound tostarch granules as well as in free, i.e. soluble form. During theexpression of E. coli, the enzyme activity of such proteins leads to anincreased phosphorylation of the glycogen synthesized within the cells.The molecular weight of these proteins lies within the range of 140-160kD if it is assessed by means of a SDS gel electrophoresis.

The present invention further relates to nucleic acid moleculescomprising a sequence with the nucleotide sequence indicated in Seq IDNo. 1, particularly the coding region indicated in Seq ID No. 1.

Nucleic acid molecules encoding a protein from potato, which in theplastids of the cells is partly granule-bound, and hybridizing to theabove-mentioned nucleic acid molecules of the invention or theircomplementary strand are also the subject matter of the presentinvention. In this context the term “hybridization” signifieshybridization under conventional hybridizing conditions, preferablyunder stringent conditions as described for example in Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2^(nd) Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). These nucleicacid molecules hybridizing with the nucleic acid molecules of theinvention may principally be derived from any desired organism (i.e.prokaryotes or eukaryotes, in particular from bacteria, fungi, alga,plants or animal organisms) comprising such nucleic acid molecules. Theyare preferably derived from monocotyledonous or dicotyledonous plants,particularly from useful plants, and particularly preferred fromstarch-storing plants.

Nucleic acid molecules hybridizing to the molecules according to theinvention may be isolated e.g. from genomic or from cDNA libraries ofvarious organisms.

Thereby, the identification and isolation of such nucleic acid moleculesmay take place by using the molecules according to the invention orparts of these molecules or, as the case may be, the reverse complementstrands of these molecules, e.g. by hybridization according to standardmethods (see e.g. Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

As a probe for hybridization e.g. nucleic acid molecules may be usedwhich exactly or basically contain the nucleotide sequence indicatedunder Seq ID No. 1 or parts thereof. The DNA fragments used ashybridization probe may also be synthetic DNA fragments which wereproduced by means of the conventional DNA synthesizing methods and thesequence of which is basically identical with that of a nucleic acidmolecule of the invention. After identifying and isolating geneshybridizing to the nucleic acid sequences according to the invention,the sequence has to be determined and the properties of the proteinsencoded by this sequence have to be analyzed.

Furthermore, the present invention relates to nucleic acid molecules thesequences of which, compared to the sequences of the above-mentionedmolecules, are degenerated due to the genetic code and which encode aprotein which in the plastids of plant cells is partly granule-bound.

Fragments, derivatives and allelic variants of the above-mentionednucleic acid molecules, which encode the above-mentioned protein arealso the subject matter of the present invention. Thereby, fragments aredescribed as parts of the nucleic acid molecules which are long enoughin order to encode the above-described protein. In this context, theterm derivative signifies that the sequences of these molecules differfrom the sequences of the above-mentioned nucleic acid molecules at oneor more positions and exhibit a high degree of homology to the sequencesof these molecules. Hereby, homology means a sequence identity of atleast 40%, in particular an identity of at least 60%, preferably of morethan 80% and still more preferably a sequence identity of more than 90%.The deviations occurring when comparing with the above-described nucleicacid molecules might have been caused by deletion, substitution,insertion or recombination.

Moreover, homology means that functional and/or structural equivalenceexists between the respective nucleic acid molecules or the proteinsthey encode. The nucleic acid molecules, which are homologous to theabove-described nucleic acid molecules and represent derivatives ofthese molecules, are generally variations of these nucleic acidmolecules, that constitute modifications which exert the same biologicalfunction. These variations may be naturally occurring variations, forexample sequences from different organisms, or mutations, whereby thesemutations may have occurred naturally or they may have been introduceddeliberately. Moreover the variations may be synthetically producedsequences. The allelic variants may be naturally occurring as well assynthetically produced variants or variants produced by recombinant DNAtechniques.

The proteins encoded by the various variants of the nucleic acidmolecules according to the invention exhibit certain commoncharacteristics. Enzyme activity, molecular weight, immunologicreactivity, conformation etc. may belong to these characteristics aswell as physical properties such as the mobility in gel electrophoresis,chromatographic characteristics, sedimentation coefficients, solubility,spectroscopic properties, stability, pH-optimum, temperature-optimumetc.

The nucleic acid molecules of the invention may principally be derivedfrom any organism expressing the described proteins. They are preferablyderived from plants, in particular from starch-synthesizing orstarch-storing plants. Cereals (such as barley, rye, oats, wheat etc.),maize, rice, pea, cassaya, potato etc. are particularly preferred. Theycan also be produced by means of synthesis methods known to the skilledperson.

The nucleic acid molecules of the invention may be DNA molecules, suchas cDNA or genomic DNA, as well as RNA molecules.

Furthermore, the invention relates to vectors, especially plasmids,cosmids, viruses, bacteriophages and other vectors common in geneticengineering, which contain the above-mentioned nucleic acid molecules ofthe invention.

In a preferred embodiment the nucleic acid molecules contained in thevectors are linked to regulatory elements that ensure the transcriptionand synthesis of a translatable RNA in prokaryotic and eukaryotic cells.

In a further embodiment the invention relates to host cells, inparticular prokaryotic or eukaryotic cells, which have been transformedand/or recombinantly manipulated by an above-mentioned nucleic acidmolecule of the invention or by a vector of the invention, as well ascells derived from such cells and containing a nucleic acid molecule ofthe invention or a vector of the invention. This is preferably abacterial cell or a plant cell.

It was now found that the protein encoded by the nucleic acid moleculesof the invention influences the starch synthesis or modification andthat changes in the amount of the protein in plant cells lead to changesin the starch metabolism of the plant, especially to the synthesis ofstarch with modified physical and chemical properties.

By providing the nucleic acid molecules of the invention it is possibleto produce plants by means of recombinant. DNA techniques synthesizing amodified starch which differs from the starch synthesized in wildtypeplants with respect to its structure and its physical and chemicalproperties. For this purpose, the nucleic acid molecules of theinvention are linked to regulatory elements, which ensure thetranscription and translation in plant cells, and they are introducedinto the plant cells.

Therefore, the present invention also relates to transgenic plant cellscontaining a nucleic acid molecule of the invention whereby the same islinked to regulatory elements which ensure the transcription in plantcells. The regulatory elements are preferably heterologous with respectto the nucleic acid molecule.

By means of methods known to the skilled person the transgenic plantcells can be regenerated to whole plants. The plants obtainable byregenerating the transgenic plant cells of the invention are also thesubject-matter of the present invention. A further subject-matter of theinvention are plants which contain the above-described transgenic plantcells. The transgenic plants may in principle be plants of any desiredspecies, i.e. they may be monocotyledonous as well as dicotyledonousplants. These are preferably useful plants, in particular starch-storingplants such as cereals (rye, barley, oats, wheat etc.), rice, maize,peas, cassaya and potatoes.

Due to the expression or the additional expression of a nucleic acidmolecule of the invention, the transgenic plant cells and plants of theinvention synthesize a starch which is modified when compared to starchfrom wildtype-plants, i.e. non-transformed plants, particularly withrespect to the viscosity of aqueous solutions of this starch and/or tothe phosphate content. The latter is generally increased in the starchof transgenic plant cells or plants, this altering the physicalproperties of the starch.

Therefore, the starch obtainable from the transgenic plant cells andplants of the invention is also the subject-matter of the presentinvention.

A further subject-matter of the present invention is a method for theproduction of a protein which is present in plant cells in granule-boundform as well as in soluble from, in which host cells of the inventionare cultivated under conditions that allow for the expression of theprotein and in which the protein is then isolated from the cultivatedcells and/or the culture medium.

Furthermore, the invention relates to proteins encoded by the nucleicacid molecules of the invention as well as to proteins obtainable by theabove-described method. These are preferably proteins encoded by nucleargenes and which are localized in the plastids. In the plastids theseenzymes are present in granule-bound as well as in free form. In an SDSgel electrbphoresis, the respective proteins from Solanum tuberosumexhibit a molecular weight of 140-160 kD and, during the expression ofE. coli, lead to an increased phosphorylation of the glycogensynthesized within the cells.

A further subject-matter of the invention are antibodies whichspecifically recognize a protein of the invention. These may bemonoclonal as well as polyclonal antibodies.

Furthermore, the present invention relates to nucleic acid moleculesspecifically hybridizing with a nucleic acid molecule of the inventionand exhibiting a length of at least 15 nucleotides. In this contextspecifically hybridizing signifies that under conventional hybridizationconditions, preferably under stringent conditions, cross-hybridizationwith sequences encoding other proteins does not significantly occur.Such nucleic acid molecules preferably have a length of at least 20,more preferably a length of at least 50 and most preferably a length ofat least 100 nucleotides. Such molecules can be used, for example, asPCR primers, as hybridization probes or as DNA molecules which encodeantisense RNA.

Furthermore, it was found that it is possible to influence theproperties of the starch synthesized in plant cells by reducing theamount of proteins encoded by the nucleic acid molecules according tothe invention in the cells. This reduction may be effected, for example,by means of antisense expression of the nucleic acid molecules of theinvention, expression of suitable ribozymes or cosuppression.

Therefore, DNA molecules encoding an antisense RNA which iscomplementary to transcripts of a DNA molecule of the invention are alsothe subject-matter of the present invention, as well as these antisensemolecules. Thereby, complementary does not signify that the encoded RNAhas to be 100% complementary. A low degree of complementarity issufficient, as long as it is high enough in order to inhibit theexpression of a protein of the invention upon expression in plant cells.The transcribed RNA is preferably at least 90% and most preferably atleast 95% complementary to the transcript of the nucleic acid moleculeof the invention. In order to cause an antisense-effect during thetranscription in plant cells such DNA molecules have a length of atleast 15 bp, preferably a length of more than 10 bp and most preferablya length of more than 500 bp, however, usually less than 5000 bp,preferably shorter than 2500 bp.

The invention further relates to DNA molecules which, during expressionin plant cells, lead to the synthesis of an RNA which in the plant cellsdue to a cosupression-effect reduces the expression of the nucleic acidmolecules of the invention encoding the described protein. The principleof the cosupression as well as the production of corresponding DNAsequences is precisely described, for example, in WO 90/12084. Such DNAmolecules preferably encode a RNA having a high degree of homology totranscripts of the nucleic acid molecules of the invention. It is,however, not absolutely necessary that the coding RNA is translatableinto a protein.

In a further embodiment the present invention relates to DNA moleculesencoding an RNA molecule with ribozyme activity which specificallycleaves transcripts of a DNA molecule of the invention as well as theseencoded RNA molecules.

Ribozymes are catalytically active RNA molecules capable of cleaving RNAmolecules and specific target sequences. By means of recombinant DNAtechniques it is possible to alter the specificity of ribozymes. Thereare various classes of ribozymes. For practical applications aiming atthe specific cleavage of the transcript of a certain gene, use ispreferably made of representatives of two different groups of ribozymes.The first group is made up of ribozymes which belong to the group Iintron ribozyme type. The second group consists of ribozymes which as acharacteristic structural feature exhibit the so-called “hammerhead”motif. The specific recognition of the target RNA molecule may bemodified by altering the sequences flanking this motif. By base pairingwith sequences in the target molecule these sequences determine theposition at which the catalytic reaction and therefore the cleavage ofthe target molecule takes place. Since the sequence requirements for anefficient cleavage are extremely low, it is in principle possible todevelop specific ribozymes for practically each desired RNA molecule.

In order to produce DNA molecules encoding a ribozyme which specificallycleaves transcripts of a DNA molecule of the invention, for example aDNA sequence encoding a catalytic domain of a ribozyme is bilaterallylinked with DNA sequences which are homologous to sequences of thetarget enzyme.

Sequences encoding the catalytic domain may for example be the catalyticdomain of the satellite DNA of the SCMo virus (Davies et al., Virology177 (1990), 216-224) or that of the satellite DNA of the TobR virus(Steinecke et al., EMBO J. 11 (1992), 1525-1530; Haseloff and Gerlach,Nature 334 (1988), 585-591). The DNA sequences flanking the catalyticdomain are preferably derived from the above-described DNA molecules ofthe invention.

In a further embodiment the present invention relates to vectorscontaining the above-described DNA molecules, in particular those inwhich the described DNA molecules are linked with regulatory elementsensuring the transcription in plant cells.

Furthermore, the present invention relates to host cells containing thedescribed DNA molecules or vectors. The host cell may be a prokaryoticcell, such as a bacterial cell, or a eukaryotic cell. The eucaryotichost cells are preferably plant cells.

Furthermore, the invention relates to transgenic plant cells containingan above-described DNA molecule encoding an antisense-RNA, a ribozyme oran RNA which leads to a cosuppression effect, whereby the DNA moleculeis linked to DNA elements ensuring the transcription in plant cells.These transgenic plant cells may be regenerated to whole plantsaccording to well-known techniques. Thus, the invention also relates toplants which may be obtained through regeneration from the describedtransgenic plant cells, as well as to plants containing the describedtransgenic plant cells. The transgenic plants themselves may be plantsof any desired plant species, preferably useful plants, particularlystarch-storing ones, as indicated above.

Due to the expression of the described DNA molecules encoding antisenseRNA, a ribozyme or a cosupression RNA in the transgenic plant cells theamount of proteins encoded by the DNA molecules of the invention whichare present in the cells in endogenic form, is reduced. Surprisingly,this reduction leads to a drastic change of the physical and chemicalproperties of the starch synthesized in the plant cells, in particularwith respect to the viscous properties of the aqueous solutions of thisstarch, to the phosphate content as well as to the release of reducingsugars in the storage of the plant cells or plant parts at lowtemperatures. The properties of the starch synthesized in the transgenicplant cells is explicitely described below.

Thus, the starch obtainable from the described transgenic plant cellsand plants is also the subject matter of the present invention.

Furthermore, the invention relates to the antisense RNA moleculesencoded by the described DNA molecules, as well as to RNA molecules withribozyme activity and RNA molecules which lead to a cosupression effectwhich are obtainable, for example, by means of transcription.

A further subject-matter of the invention is a method for the productionof transgenic plant cells, which in comparison to non-transformed cellssynthesize a modified starch. In this method the amount of proteinsencoded by the DNA molecules of the invention, which are present in thecells in endogenic form, is reduced in the plant cells.

In a preferred embodiment this reduction is effected by means of anantisense effect. For this purpose the DNA molecules of the invention orparts thereof are linked in antisense orientation with a promoterensuring the transcription in plant cells and possibly with atermination signal ensuring the termination of the transcription as wellas the polyadenylation of the transcript. In order to ensure anefficient antisense effect in the plant cells the synthesized antisenseRNA should exhibit a minimum length of 15 nucleotides, preferably of atleast 100 nucleotides and most preferably of more than 500 nucleotides.Furthermore, the DNA sequence encoding the antisense RNA should behomologous with respect to the plant species to be transformed. However,DNA sequences exhibiting a high degree of homology to DNA sequenceswhich are present in the cells in endogenic form may also be used,preferably with an homology of more than 90% and most preferably with anhomology of more than 95%.

In a further embodiment the reduction of the amount of proteins encodedby the DNA molecules of the invention is effected by a ribozyme effect.The basic effect of ribozymes as well as the construction of DNAmolecules encoding such RNA molecules have already been described above.In order to express an RNA with ribozyme activity in transgenic cellsthe above described DNA molecules encoding a ribozyme are linked withDNA elements which ensure the transcription in plant cells, particularlywith a promoter and a termination signal. The ribozymes synthesized inthe plant cells lead to the cleavage of transcripts of DNA molecules ofthe invention which are present in the plant cells in endogenic form.

A further possibility in order to reduce the amount of proteins encodedby the nucleic acid molecules of the invention is cosupression.Therefore, the plant cells obtainable by the method of the invention area further subject matter. These plant cells are characterized in thattheir amount of proteins encoded by the DNA molecules of the inventionis reduced and that in comparison to wildtype cells they synthesize amodified starch.

Furthermore, the invention relates to plants obtainable by regenerationof the described plant cells as well as to plants containing thedescribed cells of the invention.

The starch obtainable from the described plant cells and plants is alsothe subject-matter of the present invention. This starch differs fromstarch obtained from non-transformed cells or plants in its physicaland/or chemical properties. When compared to starch from wildtypeplants, the starch exhibits a reduced phosphate content. Moreover, theaqueous solutions of this starch exhibit modified viscous properties.

In a preferred embodiment the phosphate content of the described starchis reduced by at least 50%, more preferably by at least 75% and in aparticularly preferred embodiment by more than 80% in comparison tostarch derived from wildtype plants.

The modified viscosity of the aqueous solution of this starch is itsmost advantageous feature.

A well-established test for determining the viscosity is the so-calledBrabender test. This test is carried out by using an appliance which isfor example known as viscograph E. This equipment is produced and sold,among others, by Brabender fOHG Duisburg (Germany).

The test basically consists in first heating starch in the presence ofwater in order to assess when hydratization and the swelling of thestarch granules takes place. This process which is also namedgelatinization or pastification is based on the dissolving the hydrogenbonds and involves a measurable increase of the viscosity in the starchsuspension. While further heating after gelatinization leads to thecomplete dissolving of the starch particles and to a decrease ofviscosity, the immediate cooling after gelatinization typically leads toa increase in the viscosity (see FIG. 3). The result of the Brabendertest is a graph which shows the viscosity depending on time, whereby atfirst the solution is heated to above the gelatinization temperature andthen cooled.

The analysis of the Brabender graph is generally directed to determiningthe pastification temperature, the maximum viscosity during heating, theincrease in viscosity during cooling, as well as the viscosity aftercooling. These parameters are important characteristics when it comes tothe quality of a starch and the possibilty to use it for variouspurposes.

The starch which may for example be isolated from potato plants in whichthe amount of proteins of the invention within the cells was reduced bymeans of an antisense effect, showed characteristics strongly deviatingfrom the characteristics of starch isolated from wildtype plants.Compared with these it only shows a low increase in viscosity duringheating, a low maximum viscosity as well as a stronger increase inviscosity during cooling (see FIGS. 3, 4 and 5).

In a preferred embodiment the invention relates to starch, the aqueoussolutions of which exhibit the characteristic viscous propertiesdepicted in FIG. 4 or 5. Particularly under the conditions mentioned inExample 8 a for determining the viscosity with the help of a Brabenderviscosimeter, the modified starch, when compared to wildtype plants,exhibits the characteristic of only a low increase in viscosity whenheating the solution. This offers the opportunity of using the starchfor the production of highly-concentrated glues.

Moreover, after reaching maximum viscosity, there is only a low decreasein viscosity in the case of the modified starch. On the other hand theviscosity increases strongly on cooling; thus, the viscosity of modifiedstarch is higher than the viscosity of starch from wildtype plants.

By reducing the amount of proteins of the invention in transgenic plantcells it is furthermore possible to produce a starch which has theeffect that when plant parts containing this starch are stored at lowtemperatures, in particular at 4-8° C., less reducing sugars arereleased than is the case which starch from non-transformed cells. Thisproperty is particularly advantageous, for example, for providingpotatoes which during storage at low temperatures release less reducingsugars and thus exhibit a reduced cold sweetening. Such potatoes areparticularly suitable for producing French fries, crisps or similarproducts since undesirable browning-reactions (Maillard reactions) areavoided or at least strongly reduced during use.

In a particularly preferred embodiment of the present invention not onlythe synthesis of a protein of the invention is reduced in thetransformed plant cells, but moreover also the synthesis of at least onefurther enzyme involved in starch synthesis and/or modification. In thiscontext, for example, starch granule-bound starch synthases or branchingenzymes are preferred. Surprisingly, it was found that potato plants inwhich the synthesis of the proteins of the invention as well as of thebranching enzyme is reduced due to an antisense effect synthesize astarch which in its properties strongly deviates from starch of wildtypeplants.

When compared to wildtype starch, the aqueous solutions of this modifiedstarch show almost no increase in viscosity during heating or cooling(cf. FIG. 6).

Furthermore, a microscopical analysis of the starch granules before andafter heating clearly shows that, when compared to wildtype plants, thestarch granules of plants modified in such a way are not open but remainbasically unchanged in their structure. Thus, this is a starch which isresistant to the heating process. If the amylose content of this starchis determined by means of the method described in the Examples, amylosecontents of more than 50%, preferably of more than 60% and mostpreferably of more than 70% are measured for this starch. The aqueoussolutions of the starch isolated from this plants preferably show thecharacteristic viscous properties depicted in FIG. 6.

Such a highly amylose-containing starch of the invention offers a numberof advantages for various uses when compared to wildtype plants. Thus,highly amylose-containing starches have a high potential for the use infoils and films. The foils and films produced on the basis of highlyamylose-containing starches, which may be used in wide areas of thepackaging industry, have the essential advantage of being biodegradable.Apart from this use which is basically covered by classical,petrochemically produced polymers, amylose has further unique fields ofapplication which are caused by the amylose's property to form helices.The helix formed by the amylose is internally hydrophobic and externallyhydrophilic. Due to this, amylose may be used for the complexation andmolecular encapsulation of low molecular or also of high molecularsubstances. Examples therefore are:

-   -   the molecular encapsulation of vitamines and substances for the        protection against oxidation, volatilization, thermal        degradation or the transition into an aqueous environment;    -   the molecular encapsulation of aromatic substances for        increasing the solubility;    -   the molecular encapsulation of fertilizers/pesticides for        stabilization and controlled release;    -   the molecular encapsulation of medical substances for        stabilizing the dosage-control and for the controlled release of        retarding preparations.

Another important property of amylose is the fact that it is a chiralmolecule. Due to the chirality it may preferably be used afterimmobilization, e.g. on a column for separating enantiomers.

Furthermore, it was surprisingly found that starch which may be isolatedfrom potato plants in which the amount of proteins of the invention inthe cells was reduced due to an antisense effect, in combination with areduction of the proteins exhibiting the enzymatic activity of a starchgranule-bound starch synthase of the isotype I (GBSSI) exhibitscharacteristics which strongly deviate from the characteristics ofstarch which may be isolated from wildtype plants. When compared tostarch from wildtype plants, the aqueous solutions of this starch onlyshow a low increase in viscosity during heating, a low maximum viscosityas well as almost no increase in viscosity during cooling (cf. FIG. 7).If the amylose/amylopectin ratio of this starch is determined, thisstarch is characterized in that almost no amylose can be measured. Theamylose content of this starch is preferably below 5%, and mostpreferably below 2%. The starch of the invention furthermore differsfrom the known starch which may be produced in transgenic potato plantsby inhibiting the GBSSI gene solely by means of recombinant DNAtechniques. Thus, this starch shows a strong increase in viscosityduring heating. The aqueous solutions of the starch of the inventionpreferably show the characteristic viscous properties depicted in FIG.7. Particularly under the conditions for determining the viscosity bymeans of a Rapid Visco Analyser described in Example 13, the modifiedstarch has the characteristic of only exhibiting a low viscosityincrease during heating when compared to wildtype starch, but also whencompared to waxy starch. This offers the opportunity to use the starchof the invention for the production of highly-concentrated glues. Themodified starch furthermore has the property that there is only a lowdecrease of viscosity after reaching the maximum viscosity, as well asalmost no increase in viscosity during cooling.

Possibilities in order to reduce the activity of a branching enzyme inplant cells were already described, for example in WO 92/14827 and WO95/26407. The reduction of the activity of a starch granule-bound starchsynthase of the isotype I (GBSSI) may be carried out by using methodsknown to the skilled person, e.g. by means of an antisense effect. DNAsequences encoding a GBSSI from potato are for example known fromHegersberg (dissertation (1988) University of Cologne), Visser et al.(Plant Sci. 64 (1989), 185-192) or van der Leiy et al. (Mol. Gen. Genet.228 (1991), 240-240-248).

The method of the invention may in principle be used for any kind ofplant species. Monocotyledonous and dicotyledonous plants are ofinterest, in particular useful plants and preferably starch-storingplants such as cereals (rye, barley, oats, wheat etc.), rice, maize,pea, cassaya and potatoes.

Within the framework of the present invention the term “regulatory DNAelements ensuring the transcription in plant cells” are DNA regionswhich allow for the initiation or the termination of transcription inplant cells. DNA regions ensuring the initiation of transcription are inparticular promoters.

For the expression of the various above-described DNA molecules of theinvention in plants any promoter functioning in plant cells may be used.The promoter may be homologous or heterologous with respect to the usedplant species. Use may, for example, be made of the 35S promoter of thecauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812)which ensures a constitutive expression in all plant tissues and also ofthe promoter construct described in WO/9401571. However, use may also bemade of promoters which lead to an expression of subsequent sequencesonly at a point of time determined by exogenous factors (such as inWO/9307279) or in a particular tissue of the plant (see e.g. Stockhauset al., EMBO J. 8 (1989), 2245-2251). Promoters which are active in thestarch-storing parts of the plant to be transformed are preferably used.In the case of potato these parts are the potato seeds, in the case ofpotatoes the tubers. In order to transform potatoes the tuber-specificB33-promoter (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) may be usedparticularly, but not exclusively.

Apart from promoters, DNA regions initiating transcription may alsocontain DNA sequences ensuring a further increase of transcription, suchas the so-called enhancer-elements.

Furthermore, the term “regulatory DNA elements” may also comprisetermination signals which serve to correctly end the transcription andto add a poly-A-tail to the transcript which is believed to stabilizethe transcripts. Such elements are described in the literature and canbe exchanged as desired. Examples for such termination sequences are the3′-nontranslatable regions comprising the polyadenylation signal of thenopaline synthase gene (NOS gene) or the octopine synthase gene (Gielenet al., EMBO J. 8 (1989), 23-29) from agrobacteria, or the3′-nontranslatable regions of the genes of the storage proteins from soybean as well as the genes of the small subunit ofribulose-1,5-biphosphate-carboxylase (ssRUBISCO).

The introduction of the DNA molecules of the invention into plant cellsis preferably carried out using plasmids. Plasmids ensuring a stableintegration of the DNA into the plant genome are preferred.

In the examples of the present invention use is made of the binaryvector pBinAR (Höfgen and Willmitzer, Plant Sci. 66 (1990), 221-230).This vector is a derivative of the binary vector pBin19 (Bevan, Nucl.Acids Res. 12 (1984), 8711-8721), which may commercially be obtained(Clontech Laboratories, Inc. USA).

However, use may be made of any other plant transformation vector whichcan be inserted into a expression cassette and which ensures theintegration of the expression cassette into the plant genome.

In order to prepare the introduction of foreign genes in higher plants alarge number of cloning vectors are at disposal, containing areplication signal for E. coli and a marker gene for the selection oftransformed bacterial cells. Examples for selection of transformed cellsagainst cells lacking the introduced DNA.

The transformed cells grow in the usual way within the plant (see alsoMcCormick et al., Plant Cell Reports 5 (1986), 81-84). The resultingplants can be cultivated in the usual way and cross-bred with plantshaving the same transformed genetic heritage or another geneticheritage. The resulting hybrid individuals have the correspondingphenotypic properties.

Two or more generations should be grown in order to ensure whether thephenotypic feature is kept stably and whether it is transferred.Furthermore, seeds should be harvested in order to ensure that thecorresponding phenotype or other properties will remain.

Due to its properties the starch obtained from the plant cells or fromthe plants of the invention is not only suitable for the specificpurposes already mentioned herein, but also for various industrial uses.

Basically, starch can be subdivided into two major fields. One fieldcomprises the hydrolysis products of starch and the so-called nativestarches. The hydrolysis products essentially comprise glucose andglucans components obtained by enzymatic or chemical processes. They canbe used for further processes, such as fermentation and chemicalmodifications. In this context, it might be of importance that thehydrolysis process can be carried out simply and inexpensively.Currently, it is carried out substantially enzymatically usingamyloglucosidase. It is thinkable that costs might be reduced by usinglower amounts of enzymes for hydrolysis due to changes in the starchstructure, e.g. increasing the surface of the grain, improveddigestibility due to less branching or a steric structure, which limitsthe accessibility for the used enzymes.

The use of the so-called native starch which is used because of itspolymer structure can be subdivided into two further areas:

(a) Use in foodstuffs

-   -   Starch is a classic additive for various foodstuffs, in which it        essentially serves the purpose of binding aqueous additives        and/or causes an increased viscosity or an increased gel        formation. Important characteristic such vectors are pBR322, pUC        series, M13 mp series, pACYC184 etc. The desired sequence may be        integrated into the vector at a suitable restriction site. The        obtained plasmid is used for the transformation of E. coli        cells. Transformed E. coli cells are cultivated in a suitable        medium and subsequently harvested and lysed. The plasmid is        recovered by means of standard methods. As an analyzing method        for the characterization of the obtained plasmid DNA use is        generally made of restriction analysis and sequence analysis.        After each manipulation the plasmid DNA may be cleaved and the        obtained DNA fragments may be linked to other DNA sequences.

In order to introduce DNA into plant host cells a wide range oftechniques are at disposal. These techniques comprise the transformationof plant cells with T-DNA by using Agrobacterium tumefaciens orAgrobacterium rhizogenes as transformation medium, the fusion ofprotoplasts, the injection and the electroporation of DNA, theintroduction of DNA by means of the biolistic method as well as furtherpossibilities.

In the case of injection and electroporation of DNA into plant cells,there are no special demands made to the plasmids used. Simple plasmidssuch as pUC derivatives may be used. However, in case that whole plantsare to be regenerated from cells transformed in such a way, a selectablemarker gene should be present.

Depending on the method of introducing desired genes into the plantcell, further DNA sequences may be necessary. If the Ti— or Ri-plasmidis used e.g. for the transformation of the plant cell, at least theright border, more frequently, however, the right and left border of theTi- and Ri-plasmid T-DNA has to be connected to the foreign gene to beintroduced as a flanking region.

If Agrobacteria are used for transformation, the DNA which is to beintroduced must be cloned into special plasmids, namely either into anintermediate vector or into a binary vector. Due to sequences homologousto the sequences within the T-DNA, the intermediate vectors may beintegrated into the Ti- or Ri-plasmid of the Agrobacterium due tohomologous recombination. This also contains the vir-region necessaryfor the transfer of the T-DNA. Intermediate vectors cannot replicate inAgrobacteria. By means of a helper plasmid the intermediate vector maybe transferred to Agrobacterium tumefaciens (conjugation). Binaryvectors may replicate in E. coli as well as in Agrobacteria. Theycontain a selectable marker gene as well as a linker or polylinker whichis framed by the right and the left T-DNA border region. They may betransformed directly into the Agrobacteria (Holsters et al. Mol. Gen.Genet. 163 (1978), 181-187). The plasmids used for the transformation ofthe Agrobacteria further comprise a selectable marker gene, such as theNPT II gene which allows for selecting transformed bacteria. TheAgrobacterium acting as host cell should contain a plasmid carrying avir-region. The vir-region is necessary for the transfer of the T-DNAinto the plant cell. Additional T-DNA may be present. The Agrobacteriumtransformed in such a way is used for the transformation of plant cells.

The use of T-DNA for the transformation of plant cells was investigatedintensely and described sufficiently in EP 120 516; Hoekema, In: TheBinary Plant Vector System Offsetdrukkerij Kanters B. V., Alblasserdam(1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4, 1-46 and Anet al. EMBO J. 4 (1985), 277-287. Some binary vectors may already beobtained commercially, such as pBIN19 (Clontech Laboratories, Inc.,USA).

For transferring the DNA into the plant cells, plant explants maysuitably be co-cultivated with Agrobacterium tumefaciens orAgrobacterium rhizogenes. From the infected plant material (e.g. piecesof leaves, stem segments, roots, but also protoplasts orsuspension-cultivated plant cells) whole plants may then be regeneratedin a suitable medium which may contain antibiotics or biozides for theselection of transformed cells. The plants obtained in such a way maythen be examined as to whether the introduced DNA is present or not.

Once the introduced DNA has been integrated in the genome of the plantcell, it usually continues to be stable there and also remains withinthe descendants of the originally transformed cell. It usually containsa selectable marker which confers resistance against biozides or againstan antibiotic such as kanamycin, G 418, bleomycin, hygromycin orphosphinotricine etc. to the transformed plant cells. The individuallyselected marker should therefore allow for a properties are flowing andsorption behavior, swelling and pastification temperature, viscosity andthickening performance, solubility of the starch, transparency and pastestructure, heat, shear and acid resistance, tendency to retrogradation,capability of film formation, resistance to freezing/thawing,digestibility as well as the capability of complex formation with e.g.inorganic or organic ions.

(b) Use in Non-Foodstuffs

-   -   The other major field of application is the use of starch as an        adjuvant in various production processes or as an additive in        technical products. The major fields of application for the use        of starch as an adjuvant are, first of all, the paper and        cardboard industry. In this field, the starch is mainly used for        retention (holding back solids), for sizing filler and fine        particles, as solidifying substance and for dehydration. In        addition, the advantageous properties of starch with regard to        stiffness, hardness, sound, grip, gloss, smoothness, tear        strength as well as the surfaces are utilized.    -   Within the paper production process, a differentiation can be        made between four fields of application, namely surface,        coating, mass and spraying.    -   The requirements on starch with regard to surface treatment are        essentially a high degree of brightness, corresponding        viscosity, high viscosity stability, good film formation as well        as low formation of dust. When used in coating the solid        content, a corresponding viscosity, a high capability to bind as        well as a high pigment affinity play an important role. As an        additive to the mass rapid, uniform, loss-free dispersion, high        mechanical stability and complete retention in the paper pulp        are of importance. When using the starch in spraying,        corresponding content of solids, high viscosity as well as high        capability to bind are also significant.    -   A major field of application is, for instance, in the adhesive        industry, where the fields of application are subdivided into        four areas: the use as pure starch glue, the use in starch glues        prepared with special chemicals, the use of starch as an        additive to synthetic resins and polymer dispersions as well as        the use of starches as extenders for synthetic adhesives. 90% of        all starch-based adhesives are used in the production of        corrugated board, paper sacks and bags, composite materials for        paper and aluminum, boxes and wetting glue for envelopes,        stamps, etc.    -   Another possible use as adjuvant and additive is in the        production of textiles and textile care products. Within the        textile industry, a differentiation can be made between the        following four fields of application: the use of starch as a        sizing agent, i.e. as an adjuvant for smoothing and        strengthening the burring behavior for the protection against        tensile forces active in weaving as well as for the increase of        wear resistance during weaving, as an agent for textile        improvement mainly after quality-deteriorating pretreatments,        such as bleaching, dying, etc., as thickener in the production        of dye pastes for the prevention of dye diffusion and as an        additive for warping agents for sewing yarns.    -   Furthermore, starch may be used as an additive in building        materials. One example is the production of gypsum plaster        boards, in which the starch mixed in the thin plaster pastifies        with the water, diffuses at the surface of the gypsum board and        thus binds the cardboard to the board. Other fields of        application are admixing it to plaster and mineral fibers. In        ready-mixed concrete, starch may be used for the deceleration of        the sizing process.    -   Furthermore, the starch is advantageous for the production of        means for ground stabilization used for the temporary protection        of ground particles against water in artificial earth shifting.        According to state-of-the-art knowledge, combination products        consisting of starch and polymer emulsions can be considered to        have the same erosion- and encrustation-reducing effect as the        products used so far; however, they are considerably less        expensive.    -   Another field of application is the use of starch in plant        protectives for the modification of the specific properties of        these preparations. For instance, starches are used for        improving the wetting of plant protectives and fertilizers, for        the dosed release of the active ingredients, for the conversion        of liquid, volatile and/or odorous active ingredients into        microcrystalline, stable, deformable substances, for mixing        incompatible compositions and for the prolongation of the        duration of the effect due to a reduced disintegration.    -   Starch may also be used in the fields of drugs, medicine and in        the cosmetics industry. In the pharmaceutical industry, the        starch may be used as a binder for tablets or for the dilution        of the binder in capsules. Furthermore, starch is suitable as        disintegrant for tablets since, upon swallowing, it absorbs        fluid and after a short time it swells so much that the active        ingredient is released. For qualitative reasons, medicinal        flowance and dusting powders are further fields of application.        In the field of cosmetics, the starch may for example be used as        a carrier of powder additives, such as scents and salicylic        acid. A relatively extensive field of application for the starch        is toothpaste.    -   The use of starch as an additive in coal and briquettes is also        thinkable. By adding starch, coal can be quantitatively        agglomerated and/or briquetted in high quality, thus preventing        premature disintegration of the briquettes. Barbecue coal        contains between 4 and 6% added starch, calorated coal between        0.1 and 0.5%. Furthermore, the starch is suitable as a binding        agent since adding it to coal and briquette can considerably        reduce the emission of toxic substances.    -   Furthermore, the starch may be used as a flocculant in the        processing of ore and coal slurry.    -   Another field of application is the use as an additive to        process materials in casting. For various casting processes        cores produced from sands mixed with binding agents are needed.        Nowadays, the most commonly used binding agent is bentonite        mixed with modified starches, mostly swelling starches.    -   The purpose of adding starch is increased flow resistance as        well as improved binding strength. Moreover, swelling starches        may fulfill more prerequisites for the production process, such        as dispersability in cold water, rehydratisability, good        mixability in sand and high capability of binding water.    -   In the rubber industry starch may be used for improving the        technical and optical quality. Reasons for this are improved        surface gloss, grip and appearance. For this purpose, the starch        is dispersed on the sticky rubberized surfaces of rubber        substances before the cold vulcanization. It may also be used        for improving the printability of rubber.    -   Another field of application for the modified starch is the        production of leather substitutes.    -   In the plastics market the following fields of application are        emerging: the integration of products derived from starch into        the processing process (starch is only a filler, there is no        direct bond between synthetic polymer and starch) or,        alternatively, the integration of products derived from starch        into the production of polymers (starch and polymer form a        stable bond).    -   The use of the starch as a pure filler cannot compete with other        substances such as talcum. This situation is different when the        specific starch properties become effective and the property        profile of the end products is thus clearly changed. One example        is the use of starch products in the processing of thermoplastic        materials, such as polyethylene. Thereby, starch and the        synthetic polymer are combined in a ratio of 1:1 by means of        coexpression to form a ‘master batch’, from which various        products are produced by means of common techniques using        granulated polyethylene. The integration of starch in        polyethylene films may cause an increased substance permeability        in hollow bodies, improved water vapor permeability, improved        antistatic behavior, improved anti-block behavior as well as        improved printability with aqueous dyes.    -   Another possibility is the use of the starch in polyurethane        foams. Due to the adaptation of starch derivatives as well as        due to the optimization of processing techniques, it is possible        to specifically control the reaction between synthetic polymers        and the starch's hydroxy groups. The results are polyurethane        films having the following property profiles due to the use of        starch: a reduced coefficient of thermal expansion, decreased        shrinking behavior, improved pressure/tension behavior,        increased water vapor permeability without a change in water        acceptance, reduced flammability and cracking density, no drop        off of combustible parts, no halides and reduced aging.        Disadvantages that presently still exist are reduced pressure        and impact strength.    -   Product development of film is not the only option. Also solid        plastics products, such as pots, plates and bowls can be        produced by means of a starch content of more than 50%.        Furthermore, the starch/polymer mixtures offer the advantage        that they are much easier biodegradable.    -   Furthermore, due to their extreme capability to bind water,        starch graft polymers have gained utmost importance. These are        products having a backbone of starch and a side lattice of a        synthetic monomer grafted on according to the principle of        radical chain mechanism. The starch graft polymers available        nowadays are characterized by an improved binding and retaining        capability of up to 1000 g water per g starch at a high        viscosity. These super absorbers are used mainly in the hygiene        field, e.g. in products such as diapers and sheets, as well as        in the agricultural sector, e.g. in seed pellets.

What is decisive for the use of the new starch modified by recombinantDNA techniques are, on the one hand, structure, water content, proteincontent, lipid content, fiber content, ashes/phosphate content,amylose/amylopectin ratio, distribution of the relative molar mass,degree of branching, granule size and shape as well as crystallization,and on the other hand, the properties resulting in the followingfeatures: flow and sorption behavior, pastification temperature,viscosity, thickening performance, solubility, paste structure,transparency, heat, shear and acid resistance, tendency toretrogradation, capability of gel formation, resistance tofreezing/thawing, capability of complex formation, iodine binding, filmformation, adhesive strength, enzyme stability, digestibility andreactivity. The most remarkable feature is viscosity.

Moreover, the modified starch obtained from the plant cells of theinvention may be subjected to further chemical modification, which willresult in further improvement of the quality for certain of theabove-described fields of application. These chemical modifications areprincipally known to the person skilled in the art. These areparticularly modifications by means of

-   -   acid treatment    -   oxidation and    -   esterification (formation of phosphate, nitrate, sulphate,        xanthate, acetate and citrate starches. Further organic acids        may also be used for esterification.)    -   formation of starch ethers (starch alkyl ether, O-allyl ether,        hydroxylalkyl ether, O-carboxylmethyl ether, N-containing starch        ethers, S-containing starch ethers)    -   formation of branched starches    -   formation of starch graft polymers.

The invention also relates to propagation material of the plants of theinvention, such as seeds, fruits, cuttings, tubers or root stocks,wherein this propagation material contains plant cells of the invention.

Deposits

The plasmids produced and/or used within the framework of the presentinvention have been deposited at the internationally acknowledgeddeposit “Deutsche Sammlung von Mikroorganismen (DSM)” in Braunschweig,Federal Republic of Germany, according to the requirements of theBudapest treaty for international acknowledgment of microorganismdeposits for patenting (deposit number; deposition date): plasmid pBinARHyg (DSM 9505) (Oct. 20, 1994) plasmid p33-anti-BE (DSM 6146) (Aug. 20,1990) plasmid pRL2 (DSM 10225) (Sep. 04, 1995)

Used media and solutions Elution buffer: 25 mM Tris pH 8.3 250 mMglycine Dialysis buffer: 50 mM Tris-HCl pH 7.0 50 mM NaCl 2 mM EDTA 14.7mM β-mercaptoethanol 0.5 mM PMSF Protein buffer: 50 mM sodium phosphatebuffer pH 7.2 10 mM EDTA 0.5 mM PMSF 14.7 mM β-mercaptoethanol Lugolsolution: 12 g KI 6 g I₂ ad1.8 l with ddH₂O 20 × SSC: 175.3 g NaCl 88.2g sodium citrate ad1000 ml with ddH₂O ph 7.0 with 10 N NaOH 10 × MEN:200 mM MOPS 50 mM sodium acetate 10 mM EDTA pH 7.0 NSEB buffer: 0.25 Msodium phosphate buffer pH 7.2 7% SDS 1 mM EDTA 1% BSA (w/v)

DESCRIPTION OF THE FIGURES

FIG. 1 shows the plasmid p35S-anti-RL.

Plasmid Structure:

-   A=fragment A: CaMV 35S promoter, nt 6909-7437 (Franck et al., Cell    21 (1980), 285-294)-   B=fragment B: Asp718 fragment from pRL1 with a length of    approximately 1949 bp    -   Orientation relative to the promoter: anti-sense    -   The arrow indicates the direction of the open reading frame.-   C=fragment C: nt 11748-11939 of the T-DNA of Ti-plasmid pTiACH5    T-DNA (Gielen et al., EMBO J. 3 (1984), 835-846)

FIG. 2 shows the plasmid pB33-anti-RL

Plasmid structure:

-   A=fragment A: B33 promoter of the patatin gene B33 from Solanum    tuberosum (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29)-   B=fragment B: Asp718 fragment from pRL1 with a length of    approximately 1949 bp    -   Orientation relative to the promoter: anti-sense    -   The arrow indicates the direction of the open reading frame.-   C=fragment C: nt 11748-11939 of the T-DNA of Ti-plasmid pTiACH5    T-DNA (Gielen et al., EMBO J. 3 (1984), 835-846)

FIG. 3 shows a Brabender curve of a aqueous starch solution, recordedwith a Viskograph-E-type Brabender viscograph, which was isolated fromnon-transformed potato plants of the variety Désirée (see also Example8). Thereby signifying: Drehm. torque [BE] Brabender unit Temp.temperature A start of pastification B maximum degree of viscosity Cstart of the 96° C. period D start of the cooling-off period E end ofthe cooling-off period F end of the end-50° C. periodThe blue line indicates the viscosity; the red line stands fortemperature.

FIG. 4 shows a Brabender curve of a aqueous starch solution, recordedwith a Viskograph-E-type Brabender viscograph, which was isolated frompotato plants transformed with the plasmid p35S-anti-RL (see alsoExample 8). For the meaning of the abbreviations see FIG. 3.

FIG. 5 shows a Brabender curve of a aqueous solution of starch frompotatoes transformed with the plasmid pB33-anti-RL (see also Example 8),recorded with a Viskograph-E-type Brabender viscograph. For the meaningof the abbreviations see FIG. 3.

FIG. 6 shows curves of aqueous solutions of starch isolated from potatoplants (see also Example 12), which were recorded with a Rapid ViscoAnalyser. The red line stands for the temperature; the blue lines 1, 2,3 and 4 show the viscosities of the following starch solutions:

-   Line 1: starch isolated from wildtype plants,-   Line 2: starch isolated from plants in which only the branching    enzyme was inhibited (cf. Example 1 of patent application    WO92/14827),-   Line 3: starch isolated from plants in which merely the    concentration of the proteins of the invention had been reduced (cf.    Example 6).-   Line 4: starch isolated from plants which had been transformed with    the plasmid p35S-anti-RL in combination with the p35SH-anti-BE    plasmid (cf. Example 12).

FIG. 7 shows curves of aqueous solutions of starch isolated from potatoplants (see also Example 13), which were recorded with a Rapid ViscoAnalyser. The red line stands for the temperature; the blue lines 1, 2,3 and 4 show the viscosities of the following starch solutions:

-   Line 1: starch isolated from wildtype plants,-   Line 2: starch isolated from plants which had solely been    transformed with the plasmid pB33-anti-GBSSI (so-called    waxy-potato),-   Line 3: starch isolated from plants which had been solely    transformed with the plasmid p35S-anti-RL (cf. Example 6).-   Line 4: starch isolated from plants which had been transformed with    the plasmid pB33-anti-RL in combination with the plasmid    pB33-anti-GBSSI (cf. Example 13).

FIG. 8 shows the pRL2 plasmid which comprises a full-length cDNA frompotato encoding an R1 enzyme.

The Examples illustrate the invention.

1. cloning

-   -   For cloning in E. coli the vector pBluescriptSK was used.    -   For plant transformation the gene constructs were cloned into        the binary vector pBinAR (Höfgen and Willmitzer, Plant Sci. 66        (1990), 221-230) and B33-Hyg.        2. Bacterial Strains    -   For the Bluescript vector and for the pBinAR and B33-Hyg        constructs use was made of the E. coli strain DH5α (Bethesda        Research Laboratories, Gaithersburg, USA).    -   The transformation of plasmid in potato plants was carried out        by means of the Agrobacterium tumefaciens strain C58C1 pGV2260        (Deblaere et al., Nucl. Acids Res. 13 (1985), 4777:4788).        3. Transformation of Agrobacterium tumefaciens    -   The DNA transfer was carried out by means of direct        transformation according to the method of Höfgen & Willmitzer        (Nucleic Acids Res. 16 (1988), 9877). The plasmid DNA of        transformed Agrobacteria was isolated according to the method of        Birnboim & Doly (Nucleic Acids Res. 7 (1979), 1513-1523) and        electrophoretically analyzed after suitable restriction        cleavage.        4. Transformation of Potatoes    -   Ten small leaves of a sterile potato culture (Solanum        tuberosum L. cv. Désirée) injured by a scalpel were treated with        10 ml MS medium (Murashige & Skoog, Physiol. Plant. 15 (1962),        473-497) with 2% sucrose. The medium contained 50 μl of a        Agrobacterium tumefaciens overnight-culture grown under        selection. After slightly shaking it for 3-5 minutes, another        incubation took place in darkness for two days. The leaves were        subsequently put on MS medium with 1.6% glucose, 5 mg/l naphthyl        acetic acid, 0.2 mg/l benzylaminopurine, 250 mg/l claforan, 50        mg/l kanamycin or 1 mg/l hygromycin B, and 0.80% Bacto Agar for        callus induction. After a one-week incubation at 25° C. and 3000        lux the leaves were put on MS-medium with 1.6% glucose, 1.4 mg/l        zeatine ribose, 20 mg/l naphthyl acetic acid, 20 mg/l giberellic        acid, 250 mg/l claforan, 50 mg/l kanamycin or 3 mg/l hygromycin        B and 0.80% Bacto Agar for shoot induction.        5. Radioactive Marking of DNA Fragments    -   The radioactive marking of DNA fragments was carried out by        means of a DNA-Random Primer Labeling Kits by Boehringer        (Germany) according to the manufacturers instructions.        6. Northern Blot Analysis    -   RNA was isolated from leave tissue according to standard        protocols. 50 μg of the RNA was separated on an agarose gel        (1.5% agarose, 1×MEN buffer, 16.6% formaldehyde). After the gel        run the gel was briefly washed in water. The RNA was transferred        to a Hybond N type nylon membrane (Amersham, UK) with 20×SSC by        means of capillary blot. The membrane was subsequently baked in        vacuum for two hours at 80° C.    -   The membrane was prehybridized in NSEB buffer for two hours at        68° C. and subsequently hybridized overnight in NSEB buffer in        the presence of the radioactively marked probe at 68° C.        7. Plant Maintenance

Potato plants were kept in the greenhouse under the followingconditions: light period 16 hours at 25000 lux and 22° C. dark period  8hours at 15° C. atmospheric humidity 60%8. Determination of the Amylose/Amylopectin Ratio in Starch Obtainedfrom Potato Plants

-   -   Starch was isolated from potato plants according to standard        methods and the amylose/amylopectin ratio was determined        according to the method described by Hovenkamp-Hermelink et al.        (Potato Research 31 (1988) 241-246).        9. Determination of Glucose, Fructose and Sucrose

In order to determine the glucose, fructose and/or sucrose content,small pieces of potato tubers (with a diameter of approx. 10 mm) arefrozen in liquid nitrogen and subsequently extracted for 30 min at 80°C. in 0.5 ml 10 mM HEPES, pH 7.5; 80% (vol./vol.) ethanol. Thesupernatant containing the soluble components is withdrawn and thevolume is determined. The supernatant is used for determining the amountof soluble sugars. The quantitative determination of soluble glucose,fructose and sucrose is carried out in a reaction mixture with thefollowing composition: 100.0 mM imidazole/HCl, pH 6.9 1.5 mM MgCl₂ 0.5mM NADP⁺ 1.3 mM ATP 10-50 μl sample 1.0 U glucose-6-phosphatedehydrogenase from yeast

The reaction mixture is incubated at room temperature for 5 minutes. Thesubsequent determination of sugars is carried out by means of standardphotometric methods by measuring the absorption at 340 nm aftersuccessive adding of 1.0 unit of hexokinase from yeast (for determiningglucose) 1.0 unit of phosphoglucoisomerase from yeast (for determiningfructose) and 1.0 unit of invertase from yeast (for determiningsucrose).

EXAMPLE 1 The Isolation of Starch Granule-Bound Proteins from PotatoStarch

The isolation of starch granule-bound proteins from potato starch hasbeen carried out by means of electroelution in an elution appliancewhich was constructed analogous to the “Model 422 Electro Eluter”(BIORAD Laboratories Inc., USA) but had a considerably greater volume(approx. 200 ml). 25 g dried starch were dissolved in elution buffer(final volume 80 ml). The starch was derived from potatoes which producean almost amylose-free starch due to the antisense-expression of a DNAsequence encoding the starch granule-bound starch synthase I (GBSS I)from potato. The suspension was heated to 70-80° C. in a water bath.Subsequently 72.07 g urea was added (final concentration 8 M) and thevolume was filled up to 180 ml with elution buffer. The starch dissolvedduring permanent stirring and acquired a paste-like consistency. Theproteins were electroeluted from the solution overnight by means of theelution appliance (100 V; 50-60 mA). The eluted proteins were carefullyremoved from the appliance. Suspended particles were removed in a briefcentrifugation. The supernatant was dialyzed at 4° C. 2 to 3 times forone hour against dialysis buffer. Subsequently, the volume of theprotein solution was determined. The proteins were precipitated byadding ammonium sulfate (final concentration 90%), which was done duringpermanent stirring at 0° C. The precipitated proteins were pelleted bycentrifugation and resuspended in protein buffer.

EXAMPLE 2 Identification and Isolation of cDNA Sequences Encoding StarchGranule-Bound Proteins

The proteins isolated according to Example 1 were used for theproduction of polyclonal antibodies from rabbit, which specificallyrecognize starch granule-bound proteins.

By means of such antibodies a cDNA expression library was subsequentlyscreened for sequences encoding starch granule-bound proteins, usingstandard methods.

The expression library was produced as follows:

Poly (A⁺)-mRNA was isolated from potato tubers of the “Berolina”variety. Starting from the poly (A⁺)-mRNA, cDNA was produced accordingto the Gubler and Hoffmann method (Gene 25 (1983), 263-269), using anXho I-Oligo d(t)₁₈ primer. This cDNA was cut with Xho I after EcoRI-linker addition and ligated in an oriented manner in a lambda ZAP IIvector (Stratagene) cut with EcoR I and Xho I. Approximately 500,000plaques of a cDNA library constructed in such a way were screened forsequences which were recognized by polyclonal antibodies directedagainst starch granule-bound proteins.

In order to analyze the phage plaques these were transferred tonitrocellulose filters which had previously been incubated in a 10 mMIPTG solution for 30 to 60 minutes and had subsequently been dried onfilter paper. The transfer took place at 37° C. for 3 hours.Subsequently, the filters are incubated at room temperature for 30minutes in block reagent and washed for 5-10 minutes in TBST buffer. Thefilters were shaken with the polyclonal antibodies directed againststarch granule-bound proteins in a suitable dilution for one hour atroom temperature or for 16 hours at 4° C. The identification of plaquesexpressing a protein which was recognized by the polyclonal antibodieswas carried out by means of the “Blotting detection kit for rabbitantibodies RPN 23” (Amersham UK) according to the manufacturer'sinstructions.

Phage clones of the cDNA library expressing a protein which wasrecognized by the polyclonal antibodies were further purified by usingstandard methods.

By means of the in-vivo excision method, E. coli clones were obtainedfrom positive phage clones containing a double-stranded pBluescriptplasmid with the corresponding cDNA insertion. After checking the sizeand the restriction pattern of the insertions a suitable clone, pRL1,was further analyzed.

EXAMPLE 3 Sequence Analysis of the cDNA Insertion of the Plasmid pRL1

From an E. coli clone obtained according to Example 2 the plasmid pRL1was isolated and a part of the sequence of its cDNA insertion wasdetermined by standard procedures using the didesoxynucleotide method(Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467). Theinsertion has a length of about 2450 bp. A part of the nucleotidesequence as well as the amino acid sequence derived therefrom isindicated under Seq ID No. 3 and under Seq ID No. 4.

A sequence analysis and a sequence comparison with known DNA sequencesshowed that the sequence indicated under Seq ID No. 3 is new andexhibits no significant homology to DNA sequences known so far.Moreover, the sequence analysis showed that the cDNA insertion is only apartial cDNA in which a: part of the coding region at the 5′-end ismissing.

EXAMPLE 4 Identification and Isolation of a Complete cDNA Encoding aStarch Granule-Bound Protein from Solanum Tuberosum

In order to isolate a complete cDNA corresponding to the partial cDNAinsertion of the plasmid pRL1, a further cDNA library was produced. Thiswas a guard-cell-specific cDNA library from Solanum tuberosum which wasconstructed as follows:

At first epidermis fragments from leaves of “Desirée” variety potatoplants were produced essentially according to the Hedrich et al. method(Plant Physiol. 89 (1989), 148), by harvesting approximately 60 leavesof six-weeks-old potato plants kept in the greenhouse. The center nervewas removed from the leaves. The leaves were subsequently crushed in abig “Waring blender” (with a volume of 1 liter) four times in cooled,distilled H₂O on the highest level for 15 seconds each. The suspensionwas filtered through a nylon sieve with a mesh size of 220 μm (Nybolt,Zurich, Switzerland) and washed in cold distilled water several times.The suspension itself was filtered through a 220 μm nylon sieve andintensely washed with cold distilled water. The residues (epidermisfragments) were crushed in a smaller “Waring blender” (with a volume of250 ml) four times in distilled water and ice on a lower level for 15seconds each. The suspension was filtered through a 220 μm nylon sieveand washed intensely with cold distilled water. The epidermis fragments(residues) were microscopically examined for contamination by mesophylcells. If contamination occurred the crushing step was repeated in asmall “Waring blender”. The disruption of the guard cells of theepidermis fragments was carried out by means of pulverizing in liquidnitrogen in a cooled mortar for approximately two hours. In order toexamine the disruption of the guard cells, probes were regularly takenand microscopically examined. After two hours, or if a sufficiently highamount of guard cells had been disrupted, the obtained powder was filledinto a reaction tube (with a volume of 50 ml) and resuspended in onevolume GTC buffer (Chirgwin et al., Biochem. 18 (1979), 5294-5299). Thesuspension was centrifuged and the supernatant was filtered throughMiracloth (Calbiochem, La Jolla, Calif.). The filtrate was subjected toultracentrifugation for 16 hours, as described in Glisin et al.(Biochemistry 13 (1974), 2633-2637) and Mornex et al. (J. Clin. Inves.77 (1986), 1952-1961). After the centrifugation the RNA precipitate wasdissolved in 250 μl GTC buffer. The RNA was precipitated by adding 0.05volumes of 1 M acetic acid and 0.7 volumes of ethanol. The RNA wasprecipitated by centrifugation and the precipitate was washed with 3 Msodium acetate (pH 4.8) and 70% ethanol. The RNA was briefly dried anddissolved in DEPC treated water.

Poly A⁺-RNA was isolated from the isolated RNA according to standardmethods. Starting from the poly(A⁺)-mRNA, cDNA was produced according tothe Gubler and Hoffmann method (Gene 25 (1983), 263-269) by means of aXho I-oligo d(t)₁₈ primer. This cDNA was cut with Xho I after EcoRI-linker addition and ligated in an oriented manner in a lambda ZAP IIvector (Stratagene GmbH, Heidelberg, Germany) cut with EcoR I and Xho I.The packaging in phage heads was carried out using the Gigapack II Goldkit (stratagene GmbH, Heidelberg, Germany) according to themanufacturer's instructions.

From such a cDNA library phage clones hybridizing with the cDNAinsertion of the pRL1 plasmid were isolated and purified according tostandard methods. By means of the in vivo excision method E. coli cloneswere obtained from positive phage clones containing a double-strandedpBluescript plasmid with the corresponding cDNA insertion. Afterchecking the size and the restriction pattern of the insertions,suitable clones were subjected to restriction mapping and sequenceanalysis. From a suitable clone the plasmid pRL2 (DSM 10225) wasisolated which contains a complete cDNA which encodes a starchgranule-bound protein from potato.

EXAMPLE 5 Sequence Analysis of the cDNA Insertion of the pRL2 Plasmid

The nucleotide sequence of the cDNA insertion of the pRL2 plasmid wasdetermined as described in Example 3. The insertion has a length of 4856bp. The nucleotide sequence as well as the amino acid sequence derivedtherefrom is indicated in Seq ID No. 1 and/or Seq ID No. 2. In thefollowing, the corresponding gene will be called RL-gene.

EXAMPLE 6 The Construction of the Plasmid P35S-Anti-RL and theIntroduction of the Plasmid into the Genome of Potato Plants

By means of the restriction endonuclease Asp718 a DNA fragment with anapproximate length of 1800 bp was isolated from the pRL1 plasmid. Thiscorresponds to the DNA sequence indicated under Seq ID No. 3 andcontains a part of the open reading frame. The fragment was ligated intothe binary vector pBinAR cut with Asp718 (Höfgen and Willmitzer, PlantSci. 66 (1990), 221-230). This is a derivative of the binary vectorpBin19 (Bevan, Nucl. Acids Res. 12 (1984), 8711-8721). pBinAR wasconstructed as follows:

A fragment with a length of 529 bp comprising the nucleotides 6909-7437of the 35S promoter of the cauliflower-mosaic virus (Franck et al., Cell21 (1980), 285-294) was isolated from the plasmid pDH51 (Pietrzak etal., Nucl. Acids Res. 14, 5857-5868) as an EcoR I/Kpn I fragment andligated between the EcoR I and the Kpn I sites of the pBin19 polylinker.This led to the plasmid pBin19-A.

By means of the restriction-endonucleases Pvu II and Hind III a fragmentwith a length of 192 bp was isolated from the plasmid pAGV40(Herrera-Estrella et al., Nature 303, 209-213) comprising thepolyadenylation signal of gene 3 of the T-DNA of the Ti-plasmid pTiACH5(Gielen et al., EMBO J. 3, 835-846) (nucleotides 11749-11939). After theaddition of Sph I-linkers to the Pvu I site the fragment was ligatedbetween the Sph I and Hind III sites of pBin19-A. This led to plasmidpBinAR. By means of restriction and sequence analysis recombinantvectors were identified in which the DNA fragment is inserted in thevector in such a way that a part of the coding region of the cDNAinsertion from pRL1 is linked with the 35S promoter in antisenseorientation. The resulting plasmid p35S-anti-RL is shown in FIG. 1.

By inserting the cDNA fragment an expression cassette is produced whichconsists of the fragments A, B and C:

Fragment A (529 bp) contains the 35S promoter of the cauliflower-mosaicvirus (CaMV). The fragment comprises the nucleotides 6909 to 7437 of theCaMV (Franck et al., Cell 21 (1980), 285-294).

Apart from flanking regions, fragment B contains a part of theprotein-encoding areas of the cDNA insertion from plasmid pRL1. This wasisolated as an Asp718 fragment of pRL1 as described above and fused tothe 35S promoter in antisense orientation. Fragment C (192 bp) containsthe polyadenylation signal of gene 3 of the T-DNA of the Ti-plasmidpTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846).

The plasmid p35S-anti-RL has a size of approximately 12.8 kb. Theplasmid was transferred into potato plants by means ofAgrobacteria-mediated transformation, as described above. From thetransformed cells whole plants were regenerated. The transformed plantswere cultivated under greenhouse conditions.

By analyzing total RNA in a Northern Blot analysis concerning thedisappearance of the transcripts complementary to the cDNA, the successof the genetic modification of the plants was assessed. For thispurpose, total RNA was isolated from leaves of transformed plantsaccording to standard methods and subsequently separatedelectrophoretically on an agarose gel. Then it was transferred onto anylon membrane and hybridized with a radioactively labelled probe havingthe sequence indicated under Seq ID No. 1 or a part thereof. In about5-10% of the transformed plants the band indicating the specifictranscript under Seq ID No. 1 was missing in the Northern Blot analysis.The plants were used for analyzing the starch quality.

EXAMPLE 7 The Construction of the Plasmid pB33-Anti-RL and theIntroduction of the Plasmid into the Genome of Potato Plants

By means of the restriction endonuclease Asp718, a DNA fragment with anapproximate length of 1800 bp, which comprises a part of the openreading frame of the cDNA insertion was isolated from the plasmid pRL1and was ligated into the vector B33-Hyg which was cut with Asp718. Thisvector was constructed as follows:

The 35S promoter was removed from the pBinAR Hyg vector (DSM 9505) bymeans of the restriction endonucleases EcoR I and Asp718. A fragmentwith a length of about 1526 bp comprising the B33 promoter was isolatedfrom the plasmid p33-anti-BE (DSM 6146) by means of EcoR I and Asp718and inserted into the pBinAR Hyg vector (DSM 9505) cut with EcoR I andAsp718.

By inserting the cDNA fragment into the Asp718 site of the B33-Hygplasmid, an expression cassette is produced which consists of thefragments A, B and C as follows (FIG. 4):

Fragment A contains the B33 promoter from Solanum tuberosum (EP 3775092; Rocha-Sosa et al., EMBO J. 8 (1989), 23-29).

Apart from flanking regions, fragment B contains a part of the proteinencoding region of the cDNA insertion from the pRL1 plasmid. This wasisolated as an Asp718 fragment from pRL1 as described above and fused tothe B33 promoter in B33-Hyg in antisense orientation.

Fragment C (192 bp) contains the polyadenylation signal of gene 3 of theT-DNA of the Ti-plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984),835-846).

The plasmid pB33-anti-RL has a size of approximately 12.8 kb. Theplasmid was transferred into potato plants by means ofAgrobacteria-mediated transformation, as described above. From thetransformed cells whole plants were regenerated. The transformed plantswere cultivated under greenhouse conditions. By analyzing total RNA in aNorthern Blot analysis concerning the disappearance of the transcriptscomplementary to the cDNA the success of the genetic modification of theplants was assessed. For this purpose, total RNA was isolated fromtubers of transformed plants according to standard methods andsubsequently separated electrophoretically on an agarose gel. Then itwas transferred onto a nylon membrane and hybridized with aradioactively labelled probe showing the sequence indicated under Seq IDNo. 1 or a part thereof. In about 5-10% of the transformed plants theband indicating the transcript hybridizing with the cDNA of theinvention was missing in the Northern Blot Analysis. From these plantsstarch was isolated from tubers and analyzed as described in Example 8.

EXAMPLE 8 Analysis of the Transformed Potato Plants

The potato plants transformed according to Example 6 and Example 7 wereexamined with regard to the properties of the synthesized starch.Analyses were carried out with various lines of the potato plants whichhad been transformed with the plasmid p35S-anti-RL or the plasmidpB33-anti-RL and which in Northern Blot analysis had not exhibited theband indicating transcripts hybridizing to the DNA sequences of theinvention.

a) Determination of the Viscosity of Aqueous Solutions of the Starch

-   -   In order to determine the viscosity of the aqueous solutions of        the starch synthesized in transformed potato plants, starch was        isolated from tubers of plants which had been transformed with        the plasmid p35S-anti-RL or the plasmid pB33-anti-RL using        standard methods. 30 g of starch were, each taken up in 450 ml        H₂O and used for analysis in an E viscograph (Brabender OHG        Duisburg (Germany)). The appliance was used according to the        manufacturer's instructions. In order to determine the viscosity        of the aqueous solution of the starch, the starch suspension was        first heated from 50° C. to 96° C. at a speed of 3° C. per        minute. The temperature was subsequently kept at 96° C. for 30        min. The solution was then cooled from 96° C. to 50° C. at a        speed of 3° C. per minute. During the whole process the        viscosity was determined. Representative results of such        measurements are set forth in the form of graphs in FIGS. 3, 4        and 5, in which the viscosity is shown depending on time. FIG. 3        shows a typical Brabender graph for starch isolated from        wildtype-plants of the potato variety Desiree. FIGS. 4 and 5        show a typical Brabender graph for starch isolated from potato        plants which had been transformed with the plasmid p35S-anti-RL        or pB33-anti-RL. From these graphs characteristic values may be        deduced.

The characteristic Values for wildtype-plants are as follows: TABLE 1Time Torque Temperature Value [min:sec] [BE] [° C.] A  6:30  60.5 ± 17.769.9 ± 0.57 B 11:30  1838.0 ± 161.2 86.0 ± 2.1  C 15:15 1412.0 ± 18.496.0 D 45:15  526.0 ± 17.0 96.0 E 60:30 812.0 ± 8.5 50.0 F 70:45 853.0 ±5.7 50.0

-   -   The values represent the average values obtained from two        different measurements.    -   In Table 1 and the following Tables 2 and 3 the abbreviations        signify the following:        A: start of pastification        B: maximum viscosity        C: start of 96° C. period        D: start of cooling-off time        E: end of cooling-off time        F: end of the end-50° C. period

For plants which had been transformed with the plasmid p35S-anti-RL(line P2), the characteristic values are the following: TABLE 2 TimeTorque Temperature Value [min:sec] [BE] [° C.] A  6:00 50.0 69.0 B 14:00820.0 93.0 C 15:15 815.0 96.0 D 45:15 680.0 96.0 E 60:30 1150.0 50.0 F70:45 1200.0 50.0

For plants which had been transformed with the plasmid pB33-anti-RL(line P3), the characteristic values are the following: TABLE 3 TimeTorque Temperature Value [min:sec] [BE] [° C.] A 7:0 31.0 71.0 B 12:45671.0 88.3 C 15:15 662.0 96.0 D 45:15 607.0 96.0 E 60:30 1063.0 50.0 F70:45 1021.0 50.0

FIGS. 3, 4 and 5 explicitly show that the starch obtained fromtransformed plants differs from starch from wildtype plants particularlyin that the viscosity increases only very slightly during heating. Thus,during heating the maximum viscosity of the modified starch fromtransformed plants is more than 50% lower than in the case of wildtypestarch.

-   -   During cooling, on the other hand, the viscosity of the starch        isolated from transformed plants increases more than in the case        of wildtype-plants.        b) Determination of the Phosphate Content of the Starch    -   The phosphate content of the starch was determined by measuring        the amount of phosphate bound to the C-6-position of the glucose        residues. For this purpose, starch was first degraded by acid        hydrolysis and the glucose-6-phosphate content was subsequently        determined by means of an enzyme test, as described in the        following.    -   100 mg starch were incubated in 500 μl 0.7 N HCl for 4 hours at        100° C. After acid hydrolysis 10 μl of the reaction were added        to 600 μl imidazole buffer (1.00 mM imidazole, 5 mM MgCl₂, pH        6.9, 0.4 mM NAD⁺). The amount of glucose-6-phosphate in the        reaction mixture was determined by conversion with the enzyme        glucose-6-phosphate-dehydrogenase. For this purpose, 1 U        glucose-6-phosphate-dehydrogenase (from Leuconostoc        mesenteroides (Boehringer Mannheim)) was added to the reaction        mixture and the amount of produced NADH was determined by        measuring the absorption at 340 nm.

The glucose-6-phosphate content of 1 mg starch is indicated in thefollowing table for non-transformed potato plants of the variety Desireeas well as for two lines (P1 (35S-anti-RL); P2(35S-anti-RL)) oftransgenic potato plants which had been transformed with the plasmidp35S-anti-RL. TABLE 4 Plants nmol glucose-6-phosphate/mg starch %Wildtype 12.89 ± 1.34  100 P1 (35S-anti-RL) 2.25 ± 0.41 17.4 P2(35S-anti-RL) 1.25 ± 0   9.7

The following table shows the glucose-6-phosphate content per milligramstarch in potato plants which were transformed with the plasmidpB33-anti-RL, compared to starch from non-transformed plants (S.tuberosum c.v. Désirée). TABLE 5 Plants nmol glucose-6-phosphate/mgstarch % Wildtype 9.80 ± 0.68 100  7 4.50 ± 0.73 45.9 37 2.64 ± 0.9926.9 45 1.14 ± 0.44 11.6 31 1.25 ± 0.49 12.8

-   -   The plants 7, 37, 45 and 31 represent independent transformants        which had been transformed with the plasmid pB33-anti-RL. Plant        37 represents line P3 for which a Brabender graph is plotted in        FIG. 5.    -   The values show that the phosphate content of the modified        starch from transgenic potato plants is at least 50% lower when        compared to starch from wildtype plants.        c) Determination of Glucose, Fructose and Sucrose Content of        Tubers after Storage at 4° C.

Tubers of plants from various transgenic lines which had beentransformed with the antisense-construct p35S-anti-RL as well as tubersof wildtype plants were stored at 4° C. or, respectively, at 20° C. indarkness, for two months. Subsequently, the amounts of glucose, fructoseand sucrose were determined as described above. For two transgenic linesthe representative values obtained were the following: TABLE 6 GlucoseFructose Sucrose 20° C. 4° C. 20° C. 4° C. 20° C. 4° C. Wildtype 0.8455.4 0.62 52.8 8.5 13.1 cv Désirée Transgenic 1.12 6.7 0.75 7.8 7.5 10.1line 15 Transgenic 1.00 6.4 0.75 7.5 6.9 6.9 line 11The values in the table are indicated in μmol hexose or surcose/g freshweight.

-   -   From the values of Table 6 it becomes obvious that the        accumulation of reducing sugars in the tubers is considerably        lower in transgenic plants stored at 4° C. than in wildtype        plants.    -   Altogether the modified starch isolated from transgenic potato        plants resembles starch from maize-wildtype plants.

However, in comparison it has the advantage that its taste is neutraland that it is therefore more suitable for various uses in thefoodstuffs area.

EXAMPLE 9 Expression of the cDNA Insertion of the pRL2 Plasmid in E.Coli

(a) Transformation of Bacterial Cells

-   -   In order to express the cDNA insertion of the plasmid pRL2 the        cells of the E. coli strain DH5α are first transformed with the        pACAC plasmid. This plasmid contains a DNA fragment encoding the        ADP-glucose-pyrophosphorylase (AGPase) from E. coli, under the        control of the lac Z promoter. The fragment had been isolated        from the vector pEcA-15 as a DraI/HaeII fragment with a size of        about 1.7 kb (see B. Müller-Röbber (1992), dissertation, FU        Berlin) and after filling in its sticky ends it had been cloned        into a pACAC184 vector linearized with HindIII. The expression        of AGPase is to cause an increase of the glycogen synthesis in        transformed E. coli cells. The cells transformed in such a way        will in the following be named E. coli-K1-cells.    -   In order to determine the enzyme activity of the protein encoded        by the cDNA of plasmid pRL2, E. coli-K1-cells were transformed        with the pRL2 plasmid. The transformed E. coli cells which        contain the pACAC plasmid as well as the pRL2 plasmid will in        the following be named E. coli-K2-cells. The transfer of the        plasmid DNA into the bacterial cells was carried out according        to the Hanahan method (J. Mol. Biol. 166 (1983), 557-580). The        transformed E. coli cells were plated onto agar culture dishes        with the following composition:

YT medium containing 1.5% Bacto agar 50 mM sodium phosphate buffer, pH7.2   1% glucose 10 μg/ml chloramphenicol in the case of E.coli-K1-cells or 10 μg/ml chloramphenicol and

-   -   10 μg/ml ampicillin in the case of E. coli-K2-cells.    -   Escherichia coli cells of the DH5a strain which had been        transformed with the plasmid pRL2+pACAC (E. coli-K2-cells) and        also—for control—solely with the pACAC plasmid (E.        coli-K1-cells), were raised on agar plates. The formed glycogen        of the various cultures was examined with respect to the degree        of phosphorylization (at the C-6 position of the glucose        molecule), as described in the following.        (b) Isolation of Bacterial Glycogen    -   In order to isolate bacterial glycogen, the bacteria colony        which had grown after transformation was floated from each 6        agar plates (Ø135 mm) with 5 ml YT medium for each plate. The        bacterial suspension was centrifuged at 4500×g for 5 minutes.        The bacterial precipitate was resuspended in 10 ml YT medium.        Disruption of the bacteria was carried out by adding 2 volumes        of disruption medium (0.2 N NaOH; 1% SDS) and by incubation at        room temperature for 5 minutes. By adding 3 volumes of EtOH        abs., incubating at 4° C. for 30 minutes and subsequent        centrifuging at 8000×g for 15 minutes, the glycogen was        sedimented. Then the precipitate was washed with 100 ml of 70%        EtOH and again sedimented by means of a centrifugation step (10        minutes at 8000×g). The washing procedure was repeated four        times.        (c) Determination of the Total Glycogen Content    -   The isolated and sedimented glycogen was first degraded into        single glucose molecules by means of acidic hydrolysis        (dissolving of the precipitate in 2 ml 0.7 N HCl; incubation for        4 hours at 100° C.). The glucose content of the solution was        determined by means of coupled enzymatic reaction of a starch        test with a photometer (Kontron) at a wave length of 340 nm        according to the manufacturer's (Boehringer Mannheim)        instructions.

The reaction buffer contains: 100 mM MOPS, pH 7.5 10 mM MgCl₂ 2 mM EDTA0.25 mM NADP 1 mM ATP 1 U/ml glucose-6-phosphate- dehydrogenase 2 U/mlhexokinase

-   -   Die measurement was carried out at 25° C. with 10 μl glucose        solution.        (d) Determination of the Glucose-6-Phosphate Content    -   In order to determine the content of glucose molecules        phosphorylated at the C-6 position, equal amounts of glucose of        the various bacterial cultures were used. By adding the same        volumes of 0.7 N KOH to the glycogens degraded into its glucose        molecules by acidic hydrolysis (as above), the solution was        neutralized.

The reaction buffer contains: 100 mM MOPS, pH 7.5 10 mM MgCl₂ 2 mM EDTA0.25 mM NADP 2 U/ml glucose-6-phosphate- dehydrogenase

-   -   The measurement was carried out at 25° C. with 100 to 150 μl        glucose solution.        (e) Identification of an Enzyme Activity Phosphorylating        Bacterial Glycogen

The results of the determination of the phosphate content of theglycogen synthesized in the bacterial cells show that the glycogen ofthe E. coli cells, which had been transformed with the pACAC+pRL2plasmids, exhibits a 290 ±25% increased phosphorylation at the C-6position of the glucose when comparing with the control reaction (E.coli cells transformed with the pACYC plasmid) (see the followingtable). glucose-6-phosphase:glucose in E. coli cells glycogen E. coli-K11:(4600 ± 1150) E. coli-K2 1:(1570 ± 390)

-   -   The degrees of phosphorylation indicated herein are the average        value of at least 6 measurements starting from 6 independent        transformations and glycogen isolations.

EXAMPLE 10 Integration of the Plasmid p35S-Anti-RL in Combination withthe Plasmid p35SH-Anti-BE into the Genome of Potato Plants

The plasmid p35S-anti-RL was constructed as described in Example 6. Theplasmid p35SH-anti-BE was constructed as described in the applicationWO95/07355, Example 3. Both plasmids were sequentially transferred intopotato plants by means of the Agrobacterium mediated transformation asdescribed above. For this purpose, the plasmid p35SH-anti-BE was firsttransformed in potato plants. Whole plants were regenerated and selectedfor a reduced expression of the branching enzyme gene. Subsequently, theplasmid p35S-anti-RL was transformed into the transgenic plants alreadyshowing a reduced expression of the branching enzyme. From thetransformed cells transgenic plants were again regenerated and thetransformed plants were cultivated under greenhouse conditions. Byanalyzing total RNA in an RNA Blot analysis with respect to thedisappearance of the transcripts complementary to the branching enzymecDNA or the RL cDNA, the success of the genetic modification of theplants with respect to a highly reduced expression of the branchingenzyme gene as well as with respect to a highly reduced expression ofthe RL gene was assessed. For this purpose, total RNA was isolated fromleaves of transformed plants according to the described methods andsubsequently separated by means of gel electrophoresis, transferred ontoa membrane, hybridized with a radioactively labelled probe showing thesequence indicated under Seq ID No. 1 or a part thereof and thenhybridized with a radioactively labelled probe showing the sequence ofthe branching enzyme cDNA (cf. WO92/14827, Example 1) or a part thereof.In about 5%-10% of the transformed plants the band indicating thespecific transcript of the sequence indicated under Seq ID No. 1 as wellas the band indicating the specific transcript of the branching enzymecDNA (cf. WO92/14827) was missing in the RNA Blot analysis. Theseplants, which were designated R4 plants were used for analyzing thequality of the starch contained in tubers.

EXAMPLE 11 Integration of the Plasmid pB33-Anti-RL in Combination withthe Plasmid pB33-Anti-GBSSI into the Genome of Potato Plants

The plasmid pB33-anti-RL was constructed as described in Example 7. Theplasmid pB33-anti-GBSSI was constructed as follows:

The DraI/DraI fragment of the promoter region of the patatin class Igene B33 from Solanum tuberosum comprising the nucleotides −1512 to +14(Rocha-Sosa et al., EMBO J 8 (1989), 23-29) was ligated into the SmaIsite of the pUC19 plasmid. From the resulting plasmid the promoterfragment was ligated into the polylinker region of the pBin19 plasmid(Bevan, Nucleic. Acids Research 12 (1984), 8711-8721) as anEcoRI/HindIII fragment. Subsequently, the 3′ EcoRI fragment 1181 to 2511of the GBSSI gene of Solanum tuberosum (Hergersberg, dissertation(1988), University of Cologne) was ligated into the EcoRI site of theresulting plasmid.

Both plasmids were transferred sequentially into potato plants by meansof Agrobacterium mediated transformation as described in Example 10.From the transformed cells whole plants were regenerated and thetransformed plants were cultivated under greenhouse conditions. Byanalyzing the complete RNA in a RNA Blot analysis with regard to thedisappearance of the transcripts complementary to the two cDNAs, thesuccess of the genetic modification of the plants was assessed. For thispurpose, total RNA was isolated from tubers of transformed plantsaccording to standard methods and subsequently separated on agarose gelby means of gel electrophoresis, transferred onto a membrane andhybridized with a radioactively labelled probe showing the sequenceindicated under Seq ID No. 1 or a part thereof. Afterwards, the samemembrane was hybridized with a radioactively labelled probe having thesequence of the GBSSI gene or a part of this sequence (Hergersberg,dissertation (1988) University of Cologne). In about 5%-10% of thetransformed plants the band indicating the transcripts hybridizing tothe cDNA of the invention or the GBSSI cDNA were missing in the RNA Blotanalysis. From the tubers of these plants, which were designated R3plants, starch was isolated and analyzed.

EXAMPLE 12 Starch Analysis of R4 Plants

The potato plants transformed according to Example 10 were examined withrespect to the properties of the synthesized starch. The analyses werecarried out with various lines of the potato plants which had beentransformed with the plasmids p35S-anti-RL and p35SH-anti-BE and whichdid no longer—or only in extremely reduced form—show the bandsindicating transcripts hybridizing to the DNA sequences of the inventionor to the sequence of the branching cDNA in RNA Blot analysis.

a) Determination of the Viscosity of Aqueous Solutions of the Starch

-   -   In order to determine the viscosity of the aqueous solutions of        the starch synthesized in transformed potato plants, starch was        isolated from tubers of plants which had been transformed with        the plasmid p35S-anti-RL and the plasmid p35SH-anti-BE using        standard methods. 2 g of starch were each dissolved in 25 ml H₂O        and used for analysis with a Rapid Visco Analyser (Newport        Scientific Pty Ltd, Investment Support Group, Warriewood NSW        2102, Australia). The equipment was used according to the        instructions of the manufacturer. In order to determine the        viscosity of the aqueous solution of the starch, the starch        suspension was first heated from 50° C. to 95° C. with a speed        of 12° C. per minute. The temperature was then kept at 95° C.        for 2.5 minutes. Afterwards, the solution was cooled from 95° C.        to 50° C. with a speed of 12° C. per minute. During the whole        process the viscosity was measured. Representative results of        such measurements are set forth in the form of graphs in which        the viscosity is shown depending on time. FIG. 6 shows a typical        RVA graph for starch isolated from the wildtype-plants of potato        of the variety Désirée. Lines 2 and 3 show a typical RVA graph        for starch isolated from the tubers of plants which had been        transformed with the plasmid p35SH-anti-BE and with the plasmid        p35S-anti-RL, respectively. Line 4 shows a typical RVA graph for        starch isolated from tubers of plants which had been transformed        with plasmid p35SH-anti-BE in combination with plasmid        p35S-anti-RL. Line 4 is characterized in that there is no        temperature-dependent increase of viscosity.        b) Determination of the Amylose/Amylopectin Ratio    -   Starch which was isolated from the tubers of transformed potato        plants was examined with respect to the ratio of amylose to        amylopectin. The plant line R4-1 (shown in line 4 of FIG. 6)        exhibited an amylose content of more than 70%. For the plant        line R4-3 an amylose value of 27% was measured, whereas the        amylose content in wildtype starch of the Désirée variety ranges        between 19 and 22%.

EXAMPLE 13 Starch Analysis of R3 Plants

The potato plants transformed according to Example. 11 were examinedwith respect to the properties of the synthesized starch. The analyseswere carried out with various lines of the potato plants which had beentransformed with the plasmids pB33-anti-RL and pB33-anti-GBSSI and whichdid no longer—or only in extremely reduced form—show the bandsindicating transcripts hybridizing to the DNA sequences of the inventionor to the sequence of the GBSSI cDNA in RNA Blot analysis.

a) Determination of the Viscosity of Aqueous Solutions of the Starch

-   -   In order to determine the viscosity of the aqueous solution of        the starch synthesized in transformed potato plants, starch was        isolated from tubers of plants which had been transformed with        the plasmid pB33-anti-RL in combination with the plasmid        pB33-anti-GBSSI using standard methods. The viscosity was        determined by means of a Rapid Visco Analyser according to the        method described in Example 12, part a. The results are        indicated in FIG. 7. In line 1, FIG. 7 shows a typical RVA graph        for starch isolated from the wildtype-plants of the Désirée        potato variety. Lines 2 and 3 show typical RVA graphs for        starches isolated from potato plants which had been transformed        with the plasmid pB33-anti-GBSSI and with the plasmid        p35S-anti-RL, respectively. Line 4 shows a typical RVA graph for        starch isolated from potato plants which had been transformed        with the plasmid pB33-anti-GBSSI in combination with the plasmid        pB33-anti-RL. This graph is characterized in that the maximum        viscosity and the increase of viscosity at 50° C. are missing.        Furthermore, this starch is characterized in that the glue        obtained after RVA treatment exhibits almost no retrogradation        after incubating at room temperature for several days.        b) Determination of the Amylose/Amylopectin Ratio    -   Starch which was isolated from the tubers of transformed potato        plants was examined with respect to the ratio of amylose to        amylopectin. The plant line R3-5 (shown in line 4 of FIG. 7)        exhibited an amylose content of less than 4%. For the plant line        R3-6 an amylose content of less than 3% was measured. The        amylose content in wildtype starch of the Desiree variety ranges        between 19 and 22%.        c) Determination of the Phosphate Content of Starch    -   The phosphate content of the starch was determined by measuring        the amount of phosphate bound to the C-6-position of the glucose        residues. For this purpose, starch was first degraded by acid        hydrolysis and the glucose-6-phosphate content was subsequently        determined by means of an enzyme test, as described in the        following.    -   100 mg starch were incubated in 500 μl 0.7 N HCl for 4 hours at        100° C. After acid hydrolysis 10 μl of the reaction mixture were        added to 600 μl imidazole buffer (100 mM imidazole, 5 mM MgCl₂,        pH 6.9, 0.4 mM NAD⁺). The amount of glucose-6-phosphate in the        preparation is determined by conversion with the enzyme        glucose-6-phosphate-dehydrogenase. For this purpose, 1 U        glucose-6-phosphate-dehydrogenase (from Leuconostoc        mesenteroides (Boehringer Mannheim)) was added to the reaction        mixture and the amount of produced NADH was determined by        measuring the absorption at 340 nm.

The glucose-6-phosphate content per 1 mg starch is indicated in thefollowing table for non-transformed potato plants of the variety Desireeas well as for the R3-5 and the R3-6 line of transgenic potato plantswhich had been transformed with the plasmid pB33-anti-RL in combinationwith the plasmid pB33-anti-GBSSI. As a comparison, the value of thestarch from the so-called waxy potato (US2-10) which had beentransformed with the plasmid pB33-anti-GBSSI, is also indicated. TABLE 7Plants nmol glucose-6-phosphate/mg starch % Wildtype 9.80 ± 0.68 100R3-5 1.32 ± 0.10 13 R3-6 1.37 ± 0.15 14 US2-10 10.82 ± 0.42  110

1-40. (canceled)
 41. A method for the production of starch, comprisingisolating starch from a transgenic plant comprising a transgenic plantcell transformed with a nucleic acid encoding a protein that is presentin plant cells in starch granule-bound form as well as in soluble formand that is involved in the phosphorylation of starch when expressed inplants and/or that increases the phosphorylation of glycogen whenexpressed in E. coli, wherein the nucleic acid is selected from thegroup consisting of: (a) a nucleic acid comprising a nucleotide sequencethat encodes a protein having the amino acid sequence of SEQ ID NO:2,(b) a nucleic acid comprising the coding region of the nucleotidesequence of SEQ ID NO: 1, (c) a nucleic acid that hybridizes to thenucleic acid molecule of (a) or (b) under stringent conditions, (d) anucleic acid the nucleotide sequence of which is degenerate as a resultof the genetic code to a nucleotide sequence of the nucleic acidmolecule of (a), (b) or (c), (e) a nucleic acid wherein said nucleicacid molecule exhibits a sequence identity of at least 60% to a nucleicacid molecule of (a) or (b), and (f) a fragment or allelic variant ofthe nucleic acid of (a), (b), (c), (d) or (e), wherein the fragment isof sufficient length to encode a protein that is present in plant cellsin starch granule-bound form as well as in soluble form and that isinvolved in the phosphorylation of starch when expressed in plantsand/or that increases the phosphorylation of glycogen when expressed inE. coli.
 42. The method of claim 41, wherein the nucleic acid is (a).43. The method of claim 41, wherein the nucleic acid is (b).
 44. Themethod of claim 41, wherein the nucleic acid is (c).
 45. The method ofclaim 41, wherein the nucleic acid is (d).
 46. The method of claim 41,wherein the nucleic acid is (e).
 47. The method of claim 41, wherein thenucleic acid is (f).
 48. The method of claim 41, wherein the plant cellis a maize, wheat or rice plant cell.
 49. A starch obtainable from atransgenic plant comprising a transgenic plant cell transformed with anucleic acid encoding a protein that is present in plant cells in starchgranule-bound form as well as in soluble form and that encodes a proteinhaving the amino acid sequence of SEQ ID NO:2 and wherein the nucleicacid molecule is linked in sense orientation to regulatory elementsensuring transcription in plant cells.
 50. The starch of claim 49,wherein the plant cell is a maize, wheat or rice plant cell.
 51. Thestarch produced by the method of claim 41.